Oligonucleotide-anthracycline and oligonucleotide-anthracyclinone conjugates

ABSTRACT

The present invention refers to conjugates formed by a natural or modified oligonucleotide, capable of forming a triple helix with a DNA chain, linked to the aglycone moiety of an anthracycline or to an anthracyclinone via an appropriated linker; such conjugates are capable to bind selectively to specific DNA regions inhibiting their transcription and therefore the formation of the corresponding codified protein.

The present application is the national stage filing of and claimspriority to International Application No. PCT/EP97/01246, filed Mar. 12,1997 and Italian Application Ser. No. FI96A000044, filed Mar. 13, 1996.

1. FIELD OF THE INVENTION

The present invention refers to conjugates wherein a natural or modifiedoligonucleotide, capable of forming a specific triple-helix with aDNA-chain, is linked, via an appropriated linker, to the aglycone moietyof an anthracycline or to an anthracyclinone and to their use for thespecific control of gene expression.

2. STATE OF THE ART

It is known [Claude Helene et al. Biochimica et Biophysica Acta 1049,99-125 (1990); Uhlmann E. et al., Chemical Reviews, 90, 543-584 (1990)]that synthetic oligonucleotides can be used for selectively inhibitingthe expression of a gene by binding to the mRNA (antisense mechanism) orto the DNA (antigene mechanism). In the first case the oligonucleotidebinds, via hydrogen bonds, to a Watson-Crick complementary sequence,present on the mRNA-target and interfers with the synthesis of thecorresponding protein. In the second case the oligonucleotiderecognises, and binds only to a polypurine:polypyrimidine region ofdouble-stranded DNA. The synthetic oligomer, which places itself in themajor groove of the DNA target, leads to the formation of a triple-helixsegment via Hoogsteen or reverse-Hoogsteen hydrogen bonds with thepurine strand. The presence of the triple-helix structure can interferewith the replication and transcription of the gene through variousmechanisms. In order to have a permanent effect it is necessary that thestructure of the triple-helix be thermodinamically stable. Suchstability can be increased by an appropriate molecule (intercalator)added to one or both ends of the oligonucleotide [Nguyen T. Thuong etal. Angew. Chem. Int. Ed. Engl., 32, 666-690 (1993)]. Among theintercalating molecules daunorubicin was already investigated [V. F.Zarytova et al. Nucleosides and Nucleotides, 10, 575-577 (1991); U.Asseline et al. Tetrahedron, 48, 1233-1254 (1992)] but the resultsobtained are considered not satisfactory, since the increase instability of the so obtained triple-helix complex is similar to thatshown by other conjugates already studied [T.Montaney-Garestier et al.in Molecular Basis of Specificity in Nucleic Acid-Drug Interaction,Kluver Academic Publishers, p. 275-290, Nederlands (1990)]. It istherefore clear the interest of new conjugates with molecular residuethat impart a higher stability to the triple-helix structures they form.

DETAILED DESCRIPTION OF THE INVENTION

The present invention refers to new conjugates wherein a natural orsynthetically modified oligonucleotide, capable of forming a stabletriple-helix with specific DNA-regions, is covalently bound through anappropriated linker, to the aglycone moiety of an anthracycline or to ananthracyclinone. Surprisingly, this particular way of connecting theoligonucleotide to the intercalator is very efficient for obtaining thedesired results, i.e. a higher stability compared to that of conjugatesdescribed in the art.

Oligonucleotides capable of forming a triple-helix with a DNA-chain areknown and are described, for example, in: Nguyen T. Thuong et al. Angew,Chem. Int. Ed. Engl., 32, 666-690 (1993).

Among the oligonucleotides, according to the present invention theoligodeoxynucleotides (ODN) are preferred. In particular according tothe invention the following ODN can be used:

5'd(T₃ CT₂ CT₂ CT₂); SEQ ID NO.:1

5'd(T₂ GTG₂ TG₂ T₂ GTG₂); SEQ ID NO.:2

5'd(GAGA₆ (GA)₃); SEQ ID NO.:3

5'd(T₃ C^(5Me) T₂ C^(5Me) T₂ C^(5Me) T₂); SEQ ID NO.:4

5'd(TC^(5Me) ₃ T₆ C^(5Me) TC^(5Me)); SEQ ID NO.:5

5'd(TGTGT₅ GT₃ GT₂ T₄ GT₃); SEQ ID NO.:6

5'd(T₄ C^(5Me) T₄ G₆) SEQ ID NO.:7.

Also the linkers used according to the present invention are the linkerscommonly used in this field [see, for example: Nguyen T. Thuong et al.Angew. Chem. Int. Ed. Engl., 32, 666-690 (1993); D. J. Kessler et al.Nucleic Acids Res., 21, 4810-4815 (1993)].

In particular the following linkers were used:

--(CH₂)_(n) --, --(CH₂ --CH₂ --O)_(m) --, --(CH₂ --CH₂ --CH₂ --O)_(p)--, [(CH₂)₂₋₅ --NH]_(q) --, [(CH₂)₂₋₅ --S(O)₂ ]_(q) wherein n is aninteger from 4 to 30, p is an integer from 1 to 6, m and q, same ordifferent, are an integer from 2 to 9. Among useful anthracyclines,according to the invention, are, for example, the natural or syntheticanthracyclines having a free OH-group in position 4 and/or 6. Inparticular: daunorubicin, doxorubicin, carminomycin (or thecorresponding aglycones), the corresponding 5-imino derivatives and thecorresponding 3'-alfa-cyanomorpholinyl derivatives.

According to a particular embodiment of the invention theoligonucleotide is bound, via the linker, to the D-ring of theanthracycline or of the aglycone, as for instance at position 4.According to another particular embodiment of the invention theoligonucleotide is linked to the B-ring of the aglycone, as for instanceat position 6. The invention refers also to conjugates wherein theoligonucleotide capable of forming a triple-helix with a DNA chain isbound, via two linkers placed at its opposite ends, to two anthracyclinemolecules (on the aglycone moieties) or to two anthracyclinonemolecules, such anthracyclines or anthracyclinones being same ordifferent. Examples of conjugates according to the invention areschematically represented by Formula A: ##STR1## wherein:

    ______________________________________                                        Y =      --(CH.sub.2).sub.6 --                                                   --(CH.sub.2).sub.12 --                                                        --(CH.sub.2 CH.sub.2 OHCH.sub.2 CH.sub.2)--                                   --(CH.sub.2 CH.sub.2 NHCH.sub.2 CH.sub.2)--                                  ODN = 5'd(T.sub.3 CT.sub.2 CT.sub.2 CT.sub.2); SEQ ID NO.:1                    5'd(T.sub.2 GTG.sub.2 TG.sub.2 T.sub.2 GTG.sub.2 ; SEQ ID NO.:2                                         5'd(GAGA.sub.6 (GA).sub.3); SEQ ID NO.:3                                      5'd(T.sub.3 C.sup.5Me T.sub.2 C.sup.5Me                                     T.sub.2 C.sup.5Me T.sub.2; SEQ ID NO.:4                                         5'd(TC.sub.3 .sup.5Me T.sub.6 C.sup.5Me                                     TC.sup.5Me); SEQ ID NO.:5                             5'd(TGTGT.sub.5 GT.sub.3 GT.sub.2 T.sub.4 GT.sub.3); SEQ ID NO.:6                                       5'd(T.sub.4 C.sup.5Me T.sub.4 G.sub.6 ; SEQ                                 ID NO.:7                                           ______________________________________                                         ##STR2##     Wherein: R=COCH.sub.3, COCH.sub.2 OH, COCH.sub.2 OAc, C.sub.2 H.sub.5 ;     R'=H, OH, COOCH.sub.3 ; R"=H, daunosamin; R'"=H, CH.sub.3

The conjugates according to the invention are prepared, for example,following the hereinafter reported process.

First of all the halo-derivatives of the corresponding anthracyclinoneor anthracycline are prepared (see also Scheme 1).

Aglycone 1, obtained in high yields by known techniques, is treated withan excess of 1,6-diiodohexane in refluxing chloroform in the presence ofsilver oxide. Column chromatography of the obtained crude product allowsthe separation of the corresponding omega-iodohexamethylene derivatives2 and 3. Compound 3 is easily glycosylated with the protected sugar 4 inthe presence of trimethylsilyltriflate giving compound 5. According tothe invention, the oligonucleotides are prepared by solid phasesynthesis in an automatic synthesizer (Pharmacia) usingphosphoramidites, in order to obtain the corresponding 5'-thiophosphatederivatives. Coupling yields of about 96% are usually obtained. Afterdeprotection, the oligonucleotides are purified by ion-exchangepreparative chromatography. The fractions are analysed by ion-exchangeHPLC. The fractions having a purity higher than 90% are transformed intothe correspondig sodium salt and lyophilized. The above saidhalo-derivatives 2, 3, and 5 are then allowed to react with the sodiumsalt of the 5'-thiophosphate oligonucleotide in the presence of15-crown-5 in DMF/H₂ O giving the wanted final conjugates which arepurified by HPLC (see Scheme 2).

EXAMPLE 1

O-alkylation of carminomycinone

To a solution of carminomycinone [compound of formula (1) wherein:R=CO--CH₃ e R'=H] (2.64 g; 6.9 mmol) in anhydrous CHCl₃ (400 ml)1,6-diiodohexane (16.88 g; 50 mmol) and Ag₂ O (2.5 g; 11 mmol) are addedunder stirring. The mixture is refluxed for 4 days, the decrease of thestarting product is checked by TLC (CH₂ Cl₂ /acetone 98:2) and Ag₂ O (7mmol) is added on the third and fourth day. At the end the mixture isfiltered on paper filter, at room temperature, in order to remove thesilver salts and the solvent is eliminated under reduced pressure. Theresidue is treated with pentane, in order to separate the unreactedhaloderivative and the so obtained crude product is subjected tochromatography on silica gel (CH₂ Cl₂ /acetone 98:2). Two solid productsare obtained: one is red (620 mg) and the other is yellow (579 mg);their structure was determined by mass spectrometry and ¹ H and ¹³C-NMR. The red product is 4-dimethoxy-4-O-(6-iodohexyl)-carminomycinone,while the yellow product is4-dimethoxy-6-O-(6-iodohexyl)-carminomycinone.

EXAMPLE 2

Glycosidation

To a mixture of 4-dimethoxy-4-O-(6-iodohexyl)-carminomycinone (obtainedaccording to Example 1) (270 mg; 0.454 mmol) andN-allyloxy-1,4-bis-(4-nitro-benzoyl)daunosamin (obtained according toknown techniques) (541 mg; 1.01 mmol) molecular sieves 4 A (2.8 gr),treated at the flame, are added under anhydrous conditions; thereafterCH₂ Cl₂ (90 ml), Et₂ O (23 ml) and trimethylsilyltriflate (0.38 ml), at-5° C., are also added to the mixture. After 15' the reaction iscompleted. At 0° C. a 1% solution of sodium bicarbonate (25 ml) is addedand the mixture is diluted with CH₂ Cl₂ and therafter extracted with thesame solvent. The organic fractions are washed with 1% bicarbonate thenwith water up to neutrality. From the organic phase, afteranhydrification and evaporation of the solvent, the crude protected4-demethoxy-4-O-(6-iodohexyl)daunomicin is obtained (330 mg).

EXAMPLE 3

Deprotection

The product according to Example 2 (330 mg) is dissolved in methanol(144 ml) and methylene chloride; the solution is cooled at -5° C. and a0.5 M (1.8 ml) of K₂ CO₃ solution is added. The reaction course ischecked by TLC (CHCl₃ /iPrOH 95:5) and is completed in 1 h. The reactionmixture is added with 0.05 N HCl up to colour change from violet toorange, concentrated under reduced pressure at low temperature andfinally added with CH₂ Cl₂. The organic solution is washed with waterand dried, the solvent is removed under reduced pressure. The residue isdissolved in 50 ml of anhydrous CH₂ Cl₂ and Ph₃ P (8.25 mg; 0.0315mmol), Pd(O)-tetrakis (11 mg, 0.0095 mmol) and 2-methyl-butyric acid (80mg; 0.78 mmol) are added. The reaction is performed in the dark and thereaction course is cheked by TLC (CHCl₃ /MeOH/HCOOH/H₂ O65:7.5:1.5:0.5). At the end, the reaction mixture is diluted with CH₂Cl₂, washed with NaHCO₃ and H₂ O, dried and evaporated. The crudeproduct, 4-dimethoxy-4-O-(6-iodohexyl) daunomycin is purified bypreparative HPLC. The identity of the purified product is confirmed bymass spectrometry (FAB MS⁺ 724).

EXAMPLE 4

Synthesis of the 5'-phosphorothioate oligodeoxynucleotide ps^(5') -TTTCTT CTT CTT (ODN)

The synthesis is performed with an automatic synthesizer using the"phosphoroamidite" method. At the end of the ODN growth phase, afterdetritylation, a mixture of 0.1 M N,N-diisopropyl-(bis)cyanoethylphosphite in CH₃ CN (100 μl) and 0.5 M tetrazole in CH₃ CN (150 μl) isrecycled in the reaction column for 7 min., flux 1 ml/min (three times).After washing with CH₃ CN the thio-oxydation reaction with sulfur isperformed using a solution 0.1 M solution in CH₃ CN of Beaucage reagentwhich is passed through the reaction column for 40 sec., flux 1 ml/min.At the end of the synthesis the ODN linked to the solid substrate istreated with 28% NH₃ at 50° C. for 24 h. The crude product is purifiedby preparative ion-exchange chromatography (column h=12 cm; diam.=2 cm)and the fractions containing the product are analyzed by ion-exchangeHPLC. The fractions having a purity higher than 90% are pooled,transformed into the corresponding sodium salt by a DOWEX 50 WX 8^(R)resin, repeatedly treated with CHELEX^(R) resin to eliminate possibleresidues of bivalent metals, and lyophilized. The so obtained5'-thiophosphate oligodeoxynucleotide is characterized, in respect tothe unreacted ODN, by TLC, ³¹ P NMR, electronspray mass spectrometry.

EXAMPLE 5

Synthesis of conjugate (8) ##STR3##

To the sodium salt of the ODN obtained in Example 4 above (0.1 μmol)dissolved in DMF (125 μl) and H₂ O (50 μl), in the presence of15-crown-5 (13 μl), 1 mg of 4-demethoxy-4-O-(6-iodohexyl)daunomycin isadded. The reaction mixture is left at 50° C., checking the formation ofthe conjugate by reverse-phase HPLC. When the starting ODN is completelyconsumed the excess anthracycline is eliminated by column chromatographyusing reverse-phase silica gel RP-18. The crude conjugate is purified byreverse-phase HPLC and characterized by HPLC, UV and fluorescencespectroscopy. Analogously, the following products are obtained: ##STR4##Binding tests

The affinity of the conjugates according to the invention for the targetsequence in double strands (B):

    5'AGGACGAAAGAAGAAGAACTTT

    3'TCCTGCTTTCTTCTTCTTGAAA                                   (B)

is determined by UV and fluorescence spectroscopy. Since in the ODNforming triple-helix unmethylated cytidines are present, its bindingstrenght is a function of pH, therefore the stability of thetriple-helical complexes is measured at pH 5.5; 6.5; 6.8. Thermalstability values of the triple-helical complexes formed by target (B)and conjugates 6-8 (above described), conjugate 9 ##STR5## (prepared asdescribed by Asseline, U. et al. in Tetrahedron, 43, 1233-1254 (1992)),and the non-derivatized oligodeoxynucleotide (10) are compared.

    OH-TTT CTT CTT CTT                                         (10)

The dissociation is monitored measuring the variation of the absorbanceat 260 nm with increasing temperature. From the melting curves, obtainedby plotting the absorbance values at the various temperatures, the Tm(temperature of 50% dissociation of the triple helix into double helixtarget and single strand) values reported in TABLE 1 are obtained. Thetriple-helix formation between the above said ligands and the DNA-target(B) is confirmed by gel electrophoresis at 15° C., pH 5.5. The collecteddata show that, at all investigated pH values, the presence of adaunomycin-derivative covalently linked, via the hexamethylene linker,at the 5' extremity of the dodecamer gives rise to complexes which aremuch more stable (with the exception of 9) than those formed by theunderivatized ODN (10). The increase in thermal stability of thetriple-helix due to the anthracycline chromophores is high and similarfor all the conjugates 6-8. On the contrary conjugate 9 forms a muchless stable complex. This difference is particularly evident at pH 6.8,where the contribution of the ODN to the complex stability is very low;at 20° C., while the triple-helices formed by conjugates 6-8 are largelyundissociated, the one formed by conjugate 9 is almost completelydenatured. The fact that the higher stability of the complexes formed bythe conjugates according to the present invention is due to theintercalation of the aglycone moiety in the DNA-target is confimed byspectrofluorometric measurements. It is in fact known that thefluorescence of anthracyclines is lowered by their intercalation betweenDNA base pairs. The fluorescence intensity at 590 nm of the triple-helixcomplex at pH 5.5 is measured at 25° C. and compared to that obtained atthe same temperature after increasing the pH to 8.2, a condition that isnot compatible with the existence of triple-helix involving cytidines inthe third strand. The fluorescence measured at pH 8.2 is considerablyhigher (about three times) than that measured at acid pHs and comparableto that obtained at the same pH in the absence of the double-helical DNAtarget. These results show that the strong affinity for the doublestranded DNA of the conjugates according to the invention is aconsequence of the intercalation of the anthracycline chromophore in thetriple-helix complex. The behaviour of the conjugates according to theinvention as bifunctional ligands of DNA is confirmed by the fact thatthe intercalating moiety does not remain intercalated at pH values whichare not compatible with the binding-characteristics of theoligopyrimidine chain. It is therefore evident that the conjugatesobtained according to the invention represent products capable offorming more stable sequence specific complexes, as required by theantigene technology, with the target nucleic acid. It is also importantto note that the binding of an oligonucleotide (through a covalent bondof the type present in the herein described conjugates) to a moleculecapable of performing its action when intercalated in the DNA doublehelix (as for example antitumor anthracyclines agents) allows to directthe above said activity on specific regions of the genome.

This selective attack allows to hit only the genes which are causativeof disease. As the site of action of the new products is on DNA, that isalso the main cell target of these antitumor agents, the conjugatesaccording to the invention are useful to specifically direct these drugsagainst the activated oncogenes and against the proviral genome of aretrovirus, integrated in the host DNA. They represent therefore anefficient way to direct cytotoxic drug selectively on the target becauseof their stable complexation to the DNA in the specific region.

                  TABLE 1                                                         ______________________________________                                        Tm (temperature of 50% dissociation)                                               compounds pH 5.5       pH 6.5                                                                              pH 6.8                                      ______________________________________                                        6          55° C.                                                                              43° C.                                                                         31° C.                                   7 51° C. 35° C. 27° C.                                   8 55° C. 45° C. 36° C.                                   9 45° C. 25° C. 16° C.                                   10  41° C. 23° C. 13° C.                               ______________________________________                                         ##STR6##     wherein: a=I--(CH.sub.2).sub.6 --I/Ag.sub.2 O

b=TMSOTf/CH₂ Cl₂ /Et₂ O

c=K₂ CO₃ 0.5 M/MeOH

d=Pd(Ph₃ P)₄ /2-methylbutyric acid

R=COCH₃, COCH₂ OH, COCH₂ OAc, C₂ H₅

R'=H, OH, COOCH₃ ##STR7## wherein:

    ______________________________________                                        Y =      --(CH.sub.2).sub.6 --                                                   --(CH.sub.2).sub.12 --                                                        --(CH.sub.2 CH.sub.2 OHCH.sub.2 CH.sub.2)--                                   --(CH.sub.2 CH.sub.2 NHCH.sub.2 CH.sub.2)--                                  ODN = 5'g(T.sub.3 CT.sub.2 CT.sub.2 CT.sub.2); SEQ ID NO.:1                    5'd(T.sub.2 GTG.sub.2 TG.sub.2 T.sub.2 GTG.sub.2 ; SEQ ID NO.:2                                         5'd(GAGA.sub.6)GA(.sub.3); SEQ ID NO.:3                                       5'd(T.sub.3 C.sup.5Me T.sub.2 C.sup.5Me                                     T.sub.2 C.sup.5Me T.sub.2; SEQ ID NO.:4                                         5'd(TC.sub.3 .sup.5Me T.sub.6 C.sup.5Me                                     TC.sup.5Me); SEQ ID NO.:5                             5'd(TGTGT.sub.5 GT.sub.3 GT.sub.2 T.sub.4 GT.sub.3); SEQ ID NO.:6                                       5'd(T.sub.4 C.sup.5Me T.sub.4 G.sub.6 ; SEQ                                 ID NO.:7                                           ______________________________________                                    

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 7                                           - -  - - (2) INFORMATION FOR SEQ ID NO: 1:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -    (iii) HYPOTHETICAL: NO                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #1:                           - - TTTCTTCTTC TT              - #                  - #                      - #       12                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO: 2:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -    (iii) HYPOTHETICAL: NO                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #2:                           - - TTGTGGTGGT TGTGG              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 3:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -    (iii) HYPOTHETICAL: NO                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #3:                           - - GAGAAAAAAG AGAGA              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 4:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (D) OTHER INFORMATION: - #Bases C at positions 4, 7, 10                            are methy - #lated                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #4:                           - - TTTCTTCTTC TT              - #                  - #                      - #       12                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 5:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (D) OTHER INFORMATION: - #Bases at positions 2, 4, 6, 13                           and 15 - #are methylated                                        - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #5:                           - - TCTCTCTTTT TTCTC              - #                  - #                      - #    15                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 6:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -    (iii) HYPOTHETICAL: NO                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #6:                           - - TGTGTTTTTG TTTGTTTTTT GTTT          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO: 7:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -    (iii) HYPOTHETICAL: NO                                                 - -     (ix) FEATURE:                                                                  (D) OTHER INFORMATION: - #The base C at position 5 is methylate      - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #7:                           - - TTTTCTTTTG GGGGG              - #                  - #                      - #    15                                                                 __________________________________________________________________________

What is claimed is:
 1. Conjugates consisting of a natural or modifiedoligonucleotide capable of forming a triple-helix complex with a doublestranded DNA, linked via an appropriate linker, to the aglycone-moietyof an anthracycline or to an anthracyclinone.
 2. Conjugates according toclaim 1, wherein the oligonucleotide is linked via the linker, to theD-ring of the anthracycline or the anthracyclinone.
 3. Conjugatesaccording to claim 2 wherein the oligonucleotide is linked to the D-ringin position
 4. 4. Conjugates according to claim 1, wherein theoligonucleotide is linked to the B-ring of the anthracycline or theanthracyclinone.
 5. Conjugates according to claim 4 wherein theoligonucleotide is linked to the B-ring in position
 6. 6. Conjugatesaccording to claim 1 wherein the oligonucleotide is aoligodeoxynucleotide.
 7. Conjugates according to claim 6 wherein theoligonucleotide capable of forming a triple-helix is chosen from thegroup consisting of: 5'd(T₃ CT₂ CT₂ CT₂); 5'd(T₂ GTG₂ TG₂ T₂ GTG₂);5'd(GAGA₆ (GA)₃); 5'd(TGTGT₅ GT₃ GT₂ T₄ GT₃); 5'd(TC^(5Me) ₃ T₆ C^(5Me)TC^(5Me)); 5'd(T₃ C^(5Me) T₂ C^(5Me) T₂ C^(5Me) T₂); 5'd(T₄ C^(5Me) T₄G₆).
 8. Conjugates according to claim 1 wherein the linker is chosenfrom the group consisting of: --(CH₂)_(n) --, --(CH₂ --CH₂ --O)_(m) --,--(CH₂ --CH₂ --CH₂ --O)_(p) --, --[(CH₂)₂₋₅ --NH]_(q) --, [(CH₂)₂₋₅--S(O)₂ ]_(q) wherein n is an integer 4 to 30, p is an integer from 1 to6, m and q, same or different, are an integer from 2 to
 9. 9. Conjugatesaccording to claim 1 wherein the anthracycline is an anthracyclinehaving a free OH-group in position 4 and/or
 6. 10. Conjugates accordingto claim 9 wherein the anthracycline is chosen from the group consistingof: daunorubicin, doxorubicin, carminomycin, or the correspondinganthracyclinone, the corresponding 5-imino derivatives or thecorresponding 3'-alfa-cyanomorpholinyl derivatives.
 11. Conjugatesaccording to claim 2 wherein the aglycone is the aglycone of ananthracycline according to claim
 8. 12. Conjugates according to claim 2wherein the aglycone is the aglycone of an anthracycline according toclaim
 10. 13. Conjugates according to claim 1 wherein theoligonucleotide capable of forming a triple-helix is linked, via twolinkers placed at its opposed ends, to two anthracyclines molecules (onthe aglycones moieties) or to two anthracyclinone molecules, suchanthracyclines or anthracyclinones being same or different. 14.Conjugates according to claim 1, schematically represented by formula A:##STR8## wherein:

    ______________________________________                                               Y =   --(CH.sub.2).sub.6 --                                                           --(CH.sub.2).sub.12 --                                            --(CH.sub.2 CH.sub.2 OHCH.sub.2 CH.sub.2)--                                   --(CH.sub.2 CH.sub.2 NHCH.sub.2 CH.sub.2)--                                  ODN = 5'GAGA.sub.6 (GA).sub.3                                                  5'T.sub.3 CT.sub.2 CT.sub.2 CT.sub.2                                          5'T.sub.3 C.sup.5Me T.sub.2 C.sup.5Me T.sub.2 C.sup.5Me T.sub.2                           5'T.sub.2 GTG.sub.2 TG.sub.2 T.sub.2 GTG.sub.2                    5'T.sub.4 C.sup.5Me T.sub.4 G.sub.6                                           5'TC.sup.5Me .sub.3 T.sub.6 C.sup.5Me TC.sup.5Me                              5'TGTGT.sub.5 GT.sub.3 GT.sub.2 T.sub.4 GT.sub.3                           ______________________________________                                         ##STR9##     wherein: R=COCH.sub.3, COCH.sub.2 OH, COCH.sub.2 OAc, C.sub.2 H.sub.5 ;     R'=H, OH, COOCH.sub.3 ; R"=H, daunosamin; R'"=H, CH.sub.3.


15. Compound of the general formula (2): ##STR10## wherein R=COCH₃,COCH₂ OH, COCH₂ OAc, C₂ H₅ ; and R'=H, OH, COOCH₃.
 16. Compound of thegeneral formula (3): ##STR11## wherein R=COCH₃, COCH₂ OH, COCH₂ OAc, C₂H₅ ; and R'=H, OH, COOCH₃.
 17. Compound of the general formula (5):##STR12## wherein R=COCH₃, COCH₂ OH, COCH₂ OAc, C₂ H₅ ; and R'=H, OHCOOCH₃.
 18. Conjugates consisting of a natural or modifiedoligonucleotide according to claim 1 which are capable of forming atriple-helix complex with a double stranded DNA, linked via anappropriate linker selected from the group consisting of --(CH₂)--,--(CH₂ --CH₂ O)_(q), --(CH₂ --CH₂ --CH₂ --O)_(p), --[ (CH₂)₂₋₅--NH]_(q), [(CH₂)₂₋₅ --S(O)₂ ]_(q) wherein n is an integer of 4 to 30, pis an integer from 1 to 6, m and q are the same or different and are aninteger from 2 to 9 to the aglycone-moiety of an anthracycline or to ananthracyclinone.